Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

The SV40 can be dissected into multiple segments, each with a different cell type specificity

Sabine Schirm, ~ Josef Jiricny, 2 and Walter Schaffner, 1 1Institute fiir Molekularbiologie II der Universit/it Ziirich, H6nggerberg, CH-8093 Ziirich, Switzerland; 2Friedrich Miescher Institut, PO Box 2543, CH-4002 Basel, Switzerland

The SV40 enhancer is known to be active in a wide variety of tissues and species. It contains a number of sequence motifs that can be bound by protein factors and whose integrity is essential for full enhancer activity. We have individually analyzed three synthetic oligonucleotides derived from sequences present within the SV40 enhancer: two oligonucleotides contain variants of the enhancer "core" sequence (designated corePVUII and coreC) and the third represents a region containing a decanucleotide homology to the immunoglobulin promoters/enhancers {designated SPHI). The oligonucleotides were multimerized and linked to a [3-globin test gene. Transcripts of the test gene were analyzed following transient expression in 10 cell lines representing a broad spectrum of tissues. We show that each of the three short segments can individually act as an enhancer when present in multiple copies. None of these enhancers is ubiquitously active; however, each shows activity in a distinctive subpopulation of cell lines. This cell type specificity is most remarkable in the case of the two oligonucleotide segments containing the core sequences. One of these is primarily active in CV-I cells, whereas the other exhibits a cell type specificity identical to that of the entire enhancer, possibly identifying it as the most important sequence element within the native SV40 enhancer. Our data suggest that a particular cell type specificity is typical for individual enhancer segments, and that enhancers of differing specificity can be assembled from the individual sequence motifs by combining them in different patterns. [Key Words: SV40 enhancer; remote control; cell type-specific transcription; synthetic oligonucleotides]

Received December 5, 1986; revised version received and accepted January 8, 1987.

The phenomenon of remote transcriptional control by transcription when introduced into frog kidney (R. cis-acting DNA elements was originally discovered in Miller and W. Schaffner, unpubl.)or even algal cells mammalian cells and described as the enhancer effect (Neuhaus et al. 1984). The sequences important for SV40 (Banerji et al. 1981; Moreau et al. 1981). The first en- enhancer activity have been investigated by studying hancing DNA sequence identified was that from the deletion-duplication events (de Villiers et al. 1984; simian 40 (SV40) . When linked to homolo- Swimmer and Shenk 1984; Weber et al. 1984; Herr and gous and heterologous promoters, it was found to stimu- Gluzman 1985; Herr and Clarke 1986)and, in a more late transcription in an orientation-independent refined way, by site-directed mutagenesis (Zenke et al. manner, even from a position downstream of the gene 1986; see also Weiher et al. 1983). These studies sup- (Banerji et al. 1981). Following this discovery, a great ported our suggestion that enhancers generally represent number of viral and cellular enhancers have been re- a modular arrangement of short sequence motifs (Bos- ported. However, the SV40 enhancer is the best charac- hart et al. 1985; Dorsch-H/isler et al. 1985; Serfling et al. terized and the most often used example of a prototype 1985), each interacting with a specific cellular transcrip- enhancer. In contrast to enhancers which exhibit a strict tion factor. The first indication for the relevance of cel- cell type specificity (Banerji et al. 1983; Gillies et al. lular trans-acting factors to SV40 enhancer function 1983; for review, see Gluzman 1985; Serfling et al. 1985) came from in vivo competition studies (Sch61er and or, at least, a cell preference (de Villiers and Schaffner Gruss 1984). Cellular protein factors physically inter- 1981; Laimins et al. 1982), the SV40 enhancer is known acting in vitro with the SV40 enhancer were subse- to act in a wide variety of tissues and hosts. In addition quently detected by DNase I footprinting (Davidson et to its activity in mammalian cells, it also stimulates al. 1986; Wildeman et al. 1986). Additionally, Davidson

GENES & DEVELOPMENT 1:65-74 (1987) © by Cold Spring Harbor Laboratory ISSN 0890-9369/87 $1.00 65 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Schirm et al. et al. (1986)provided evidence for a cell type-specific order to analyze their enhancing activity individually binding of certain protein factors to overlapping sets of and to investigate their cell type specificity (Fig. 1A). For sequence motifs within the SV40 enhancer. Evidence for the choice of these segments, we took into consideration the binding of more than one factor to a given enhancer the reports by Herr and colleagues (1985, 1986)and espe- was also indirectly shown by Sen and Baltimore (1986), cially that of Zenke et al. (1986), which showed that the who found that different segments of the immunoglob- integrity of each of these regions is essential for full en- ulin kappa enhancer did not compete with each other in hancer activity. Three segments, designated corePVUII, protein-binding studies. coreC, and SPHI, were synthesized as the double- Hints that a segment of an enhancer may have a cell stranded oligonucleotides shown in Figure lB. The type specificity different from the complete enhancer corePVUII-oligonucleotide was synthesized as a 22-bp first came from studies of SV40 enhancer variants, as dimer of an l l-bp sequence. The boundaries were well as from analyses of enhancers within the polyoma- chosen in such a way that its multimerization did not virus and Herpesvirus saimiri (de Villiers and create a new junction but rather resulted in a circular Schaffner 1981; Schirm et al. 1985). An SV40 enhancer redundancy of sequences naturally occurring in the deletion variant that had duplicated a residual enhancer SV40 enhancer. Thus, the 22-bp dimer of this DNA seg- segment restored enough activity for growth in CV-1 ment contains a continuous 14-bp sequence from the ~ kidney epithelial cells (Weber et al. 1984). However, SV40 enhancer, and the stretch of alternating purines when tested in a wider variety of primate cells, this du- and pyrimidines {bp 258-265)is conserved (Fig. 1C, part plicated enhancer variant was active only in CV-1 and in a). Both the coreC- and the corePVUII-oligonucleotides lymphoid cells (M. Pettersson and W. Schaffner, un- include the "core" consensus sequence (Weiher et al. publ.). An altered cell type-specificity of a mutant SV40 1983). The monomer of the corePVUII-oligonucleotide enhancer was also quoted in Herr and Clarke (1986). In contains no other sequences besides this homologous addition, a small segment of the enhancer of Herpes- stretch (Fig. 1C, part b). In an initial experiment these virus saimiri has a pronounced cell type specificity dis- oligonucleotides were ligated to generate multiple tinct from that of the complete enhancer (S. Schirm and copies, and then inserted into the Sinai site of the vector W. Schaffner, unpubl.). A weak but significant cell type pSVXB (Tognoni et al. 1985), upstream of the SV40 early preference had also been observed for each half of the and T- gene. Plasmid was prepared polyomavirus enhancer when tested separately (Her- from pools of bacterial clones (one pool for each of the bomel et al. 1984; see also Amati 1985). three oligonucleotides)and transfected into different cell For our analysis of small segments of the SV40 en- lines. Enhancer activity of the polymerized synthetic se- hancer, we have exploited the observation that a high quences was monitored by staining for T-antigen with activity can result from multiple tandem copies of a indirect immunofluorescence. In this assay the coreC- subfunctional enhancer segment (de Villiers et al. 1984; oligonucleotide was found to be active in all cell lines Laimins et al. 1984; Weber et al. 1984) or even a short tested, namely CV-1 {monkey kidney), HeLa { oligonucleotide (Veldman et al. 1985; our own data; B. cervix carcinoma), Molt-4 (human T cells), and 3T6 Ondek and W. Herr, pets. comm.). We report that short (mouse fibroblasts), while the activity of corePVUII sequences dissected from the SV40 enhancer each have a and SPHI were restricted to CV-1 and lymphoid cells, re- characteristic cell type specificity, which can be distinct spectively (data not shown). To follow up these prelim- from that of the complete enhancer. Furthermore, we inary observations in more detail, defined multimers show that these specificities can be different even be- (corePVUII, eight copies; coreC, four and eight copies; tween two segments containing motifs of high sequence SPHI, eight copies)were inserted 1.2 kb downstream of a homology. Our findings indicate that enhancers are rabbit f~-globin test gene in plasmid JK- (Fig:. 2A). composed of multiple sequence elements which, in prin- A total of 10 different cell lines were transfected with ciple, can substitute for each other, but in a cell type- these constructs, using either the calcium phosphate specific manner. It also appears that nucleotides next to method (Graham and van der Eb 1973; Wigler et al. a characteristic enhancer motif can modulate the cell 1978) or the DEAE-dextran method (Luthman and Mag- type specificity of the motif. In addition, the finding of nusson 1983), each with modifications according to the cell type-specific activity of multimerized segments de- requirements of the cell line (see Materials and rived from an enhancer that as a whole is active in most methods; for the origins of cell lines see Table 1). In all cells suggests a complex interplay of enhancer sequence transfection experiments a negative control and a posi- motifs and their cognate factors. tive control were included (the con- taming no enhancer or the complete SV40 enhancer, re- spectively). A truncated rabbit f~-globin gene under con- Results trol of the SV40 enhancer was used as an intemal control for transfection efficiency (Picard and Schaffner 1985). Short oligonucleotides from the SV40 enhancer have Enhancer activity was quantitated by S 1 nuclease map- enhancing activity by themselves ping of RNA from transfected cells. As a minimum, Three short segments of the SV40 enhancer that can in- every cell line was transfected twice in independent ex- teract with protein factors in the context of the com- periments. The lymphoid cells each were tested three plete enhancer (Wildeman et al. 1986)were dissected in times, while the melanoma cell line Bowes and the rat

66 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Cell-type-specific SV40 enhancer segments

A earl

late ~,~ / Oorigin

72

I corePVUll coreC SPHI --~ ' ~ f" I I ~ I :TT•T•A•••••AAA•AA••AG•TGT••AAT•TGT•T•AGTTAG••T•TG•AAA•T••••A•G•T••••AG•AGG•AGAAGTAT••AAAG•ATGCAT•T•AATTAGT••AG•AA••A 294" " 2~o "Pvu[li-' 2!6o, I " 24o" I d~o 'Sphl2(;o 1 " 179

B C 280 250 coreC 5' TAGGGTGTGGAAAGTCCCCA 3' a) sv4o wt AGAACCAGCTGTGGAATGTGTGTCAGTTAGG 3' ATCCCACACCTTTCAGGGGT 5' corePVUII TGTGGAATGTGTGTGGAATGTG repeat (llbp) SPHI 5' GCAGAAGTATGCAAAGCATGCATCT 3' 3' TTCATACGTTTCGTACGTAGACGTC 5' ~A b) core consensus GTGGTTTG coreC TAGGGTGTGGAAAGTCCCCA

corePVUII 5' TGTGGAATGTGTGTGGAATGTG 3' corePVUII TGTGGAATGTGTGTGGAATGTG , ~ , , 3' CTTACACACACCTTACACACAC5' repeat " Figure 1. (A) Diagram of the SV40 regulatory region. The direction of late and early transcription, the , and the 21/22-bp promoter repeats are indicated. The enhancer is marked by a thick line; the nucleotide sequence of one 72-bp repeat and flanking sequences extending to the KpnI site at nucleotide position 295 are shown (numbering according to Buchman 1981). Three synthetic oligonucleotides derived from the SV40 enhancer sequence were investigated (boxed). (B) Synthetic oligonucleotides. The sequences were synthesized as complementary double strands, either with blunt ends (coreC) or with a 5' overhang of 4 bp (SPHI and corePVUII). {C) Comparison of core consensus sequence motifs. (Part a)The corePVUII-oligonucleotide dimer is lined up underneath sequences of the SV40 enhancer. The dimer represents a continuous stretch of 14 bp from the wild-type enhancer (indicated by stars; for further explanation see text). (Part b) The homology between the coreC- and the core PVUII-oligonucleotides is indicated by stars. Note that the core consensus sequence (Weiher et al. 1983; on top) is fully contained in both oligonucleotides.

pituitary cell line GH3 B6 both were tested four times. four copies (see Materials and methods). Though this In all cases the results were highly reproducible. changes the spacing between copies 4 and 5, we consider it unlikely that these extra four nucleotides contributed significantly to the enhancer effect with coreC8x. The The coreC-oligonucleotide is active in 9 out of 10 cell enhancer effect with eight copies of coreC was at least as lines, as is the complete SV40 enhancer high as that of the whole SV40 enhancer, irrespective of In nine of the cell lines screened, the SV40 coreC-oli- the cell type and with only one exception {Figs. 3 and 4, gonucleotide (20 bp; Fig. 1B)showed activity when lanes coreC8x and SV40 enh; Table 1). Surprisingly, in present in four or eight copies. Interestingly, eight copies CV-1 cells, the cell line commonly used for lytic growth were considerably more than twice as active as four of SV40, eight copies of coreC were only about one-sixth copies (Figs. 3 and 4, lanes coreC4x and coreC8x). Be- as active as the SV40 enhancer as determined by densito- cause coreC8x was created by dimerizing coreC4x, there metric scanning of the autoradiographs from two inde- are an extra four nucleotides between the two blocks of pendent experiments.

GENES & DEVELOPMENT 67 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Schirm et al.

A ~ /-coreC These findings confirm and extend those of A. Belayew ~~SZSI~ cOR [ / SPHI 8copies and A.T. Truong (pets. comm.) and Camper et al. (1985}, /~ "~ / core PVU II who have not seen any activity of the complete SV40 / v,..1 )¢4.. SV40enhancer ( 196 bp) enhancer in these cells when assaying for expression of ( j K_ ^"u" / ~-polylinker sequences (26bp) bacterial chloramphenicol acetyltransferase (CAT; Gorman et al. 1982). However, this effect is highly spe- Kpn I ~ 7~1- Kpn I cific, and the coreC-oligonucleotide is not generally in- active in rodent cells, since the multimerized coreC-oli- gonucleotide is active in mouse 3T6 cells when tested by immunofluorescence for T antigen (see above). For studies of enhancer activity in GH3 B6 cells, the Rous virus enhancer (Weber and Schaffner 1985)and I~N '-" B ~ "C= ~==--°~ BamHl Bgll] Asp 718 _rr the human enhancer (Boshart et al. "~:(n°"~.~'° (~ '

Figure 2. (A) Structure of the recombinants analyzed in tran- sient assays. Various DNA fragments were inserted into the XhoI site of plasmid JK-, 1.2 kb downstream of the rabbit ~- globin gene. The activity of the coreC-, SPHI-, and corePVUII- oligonucleotides leach multimerized to eight copies)was com- Table 1. Activity of DNA segments dissected from the pared with that of the SV40 enhancer (bp 100-295) and to a SV40 enhancer construct containing only polylinker sequences instead of an Cell enhancer. IB) Modified polylinker sequence of pUC18BA con- Lines Origin SPHI coreC corePVUII taining an additional restriction site for BglII and XhoI as com- pared with pUC18 (Yanisch-Perron et al. 1985). (C) Map of the CV- 1 monkey kidney {+ ) 0.17 0.17 internal reference gene clone containing the human cytomega- HeLa human cervix -- 2.8 lovirus enhancer. carcinoma MCF7 human breast ~ 2.1 carcinoma LN-18 human glioma (+) 1.1 (+) Bowes human melanoma -- 2.5 Only a moderate enhancer effect was observed in BJA-B human B-cell 0.32 2.1 Bowes melanoma cells (Fig. 3E); the SV40 enhancer and Namalwa human B-cell 1.4 1.2 -- eight copies of the coreC-oligonucleotide stimulated Molt-4 human T-cell 0.64 1.5 D 1112 hybridoma -- 1.4 transcription about 7-fold and 15-fold, respectively. In (human B-cell/ these cells, globin expression is quite high even in the spleen) absence of any enhancer, perhaps due to abundance of GH3 B6 rat pituitary ~ ~ transcription factors interacting with the globin pro- moter. However, we cannot exclude the possibility that Relative enhancer activity was determined quantitatively by efficient transcription of the enhancerless ~-globin gene densitometric scanning of the autoradiograms. The stimulation of transcription by the complete enhancer, relative to the refer- is the result of intracellular recombination with the ence gene, is referred to as 1. Numeric representations of the SV40 enhancer-containing reference plasmid. activation by eight copies of the SPHI-, coreC-, and corePVUII- Interestingly, and in contrast to all the other cell lines, oligonucleotides are given relative to that of the SV40 enhancer the rat pituitary cell line GH3 B6 was negative for both and represent the average from two independent experiments. the complete SV40 enhancer and the coreC-oligonucleo- In cases of marginal (+} or no enhancer activity we relied on tide (Fig. 5, lanes coreC4x, coreC8x and SV40 enh). visual inspection of the autoradiograms.

68 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Cell-type-specific SV40 enhancer segments

CV- 1 HeLa MCF7 LN-18 Bowes

.,. _ ~+,,.+ ebt<,'¢'

',,''.o oovo"co M <.°Cc~o°o°o~a¢5 M ¢."CoXo"oUo'aCS\(b M ~:r-~oUo°o°bo~\O~ M coXoOoOoOco \c~ M

i i : i

Ct-~ :~, /i! ...... :...... : ~ it-*

f

ref-*-- : •- ~ "" ...... --~,,.'

.....

A B C D E Figure 3. Quantitative S1 mapping of rabbit ~-globin RNA. The constructs containing multiple copies of oligonucleotides derived from the sequence of the SV40 enhancer (Fig. 2A)were transfected into different cell lines: CV-1 (A); HeLa (B); MCF7 (C); LN-18 (D); Bowes (E). In each panel the constructs investigated are arranged in the same order: (lane no enh) negative control plasmid without enhancer; (lane SPHI) eight copies of SPHI-oligonucleotide; (lane coreC4x) four copies of the coreC-oligonucleotide; (lane coreC8x) eight copies of the coreC-oligonucleotide; (lane corePVUII) eight copies of the corePVUII-oligonucleotide; (lane SV40 enh) complete SV40 enhancer; (lane Ig enh) 700-bp fragment (XbaI-EcoRI) of the mouse immunoglobulin heavy-chain enhancer (T. Gerster and W. Schaffner, unpubl.); (ct) correct terminus (354 nucleotides); (it) incorrect terminus (this band apparently results from initiation up- stream of the rabbit B-globin cap site, followed by splicing into a cryptic 3' splice site at position + 48; see de Villiers et al. 1982); (ref) truncated rabbit B-globin gene with SV40 enhancer (Picard and Schaffner 1985); (M) marker DNA fragments (plasmid pBR322 cleaved with HpalI).

Activity of the corePVUII-oligonucleotide is restricted the experiments described below were performed with a to CV-1 cells construct in which one of the dimers was inverted in The corePVUII-oligonucleotide (Fig. 1B; synthesized as a orientation with respect to the others (the same cell type 22-bp dimer of an l l-bp sequence)was further multi- specificity was observed with a recent construct having merized eight times to give a length similar to the eight all the monomers oriented head-to-tail; not shown}. The copies of the other oligonucleotides. Due to sequence re- multimerized corePVUII-oligonucleotide was highly ac- dundancy the dimer contains a 14-bp-long stretch of tive in CV-1 cells. However, no enhancer effect could be SV40 sequences (Fig. 1 C, part a). It should be noted that observed in any of the other cells lines, with the excep-

B J A- B Namalwa Namalwa Molt-4 Dl112

,,.+o,+ ,~~ + +~, .+~. ,&~,

.. %q'2~°2,~ '..t% ¢'°;4~ o° o° o°~o"~X. °; M e"co" ou ou o" co" M ~',°co~ o°"o°'o°'¢o"~ M g i ~!? ::

...... O

ct -~ ...... e it-* ..... -- !

;% m 'w e

w~ .... ~ ...... =g g ref ~ ...... ° 4 ~.~ i A Bl B2 C D Figure 4. Quantitative S1 mapping of B-globin-specific RNA in lymphoid cells. The cell lines used in the study are: BJA-B (A); Namalwa (B1 and B2){the two panels represent different exposures of the same gel to show the reference gene); Molt-4 (C); D1112 (D). The labeling of the lanes is the same as the Fig. 3; (fl) full-length probe (453 nucleotides).

GENES & DEVELOPMENT 69 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Schirm et al.

GH3 B6 motif (also called octamer motif by some authors) is found in some housekeeping gene promoters (Harvey et al. 1982; Mattaj et al. 1985; Sive et al. 1986). No activity of the multimerized SPHI-oligonucleotide could be ob- served in HeLa cells (Fig. 3B, lane SPHI)despite the func- ID tional importance of this region within the native SV40 fl ----- Ip enhancer (Zenke et al, 1986). However, this DNA seg-

Ct .--- ment has an activity pattern identical to that of the lym- it----- ~ ii phoid cell-specific immunoglobulin heavy chain (IgH) enhancer (Banerji et al. 1983; Gillies et al. 1983; Neu- O berger et al. 1983): Both the multimerized SPHI-oligonu- o cleotide of the SV40 enhancer and the mouse IgH en- hancer were active in BJA-B, Namalwa, and Molt-4 cells o (Fig. 4A, B, and C, lanes SPHI and Ig enh, and data not ref'" ~ ~:"*~u'~ ~ Ill shown), whereas in D1112 cells, a cell line of lymphoid ii origin nonpermissive for the IgH enhancer, the SPHI- segment was inactive (Fig. 4D, lanes SPHI and not Figure 5. S1 mapping of B-globin-specific RNA in rat pituitary shown). cells. The transfection of rat pituitary cells GH3 B6 was done A short segment of the IgH enhancer containing the with the same constructs as described in Fig. 3. (Lane RSV enh) decanucleotide sequence parallels the tissue,specific ac- Construct with the RSV enhancer downstream of the rabbit B- globin gene (Weber and Schaffner 1985); (fl)full-length probe tivity of the complete IgH enhancer (T. Gerster and W. (453 nucleotides); (ref) truncated J3-globin gene with human cy- Schaffner, unpubl.; data not shown). This fragment tomegalovirus enhancer. therefore behaves similarly to the SPHI-oligonucleotide in this regard. Interestingly, in a band-shift assay (Strauss and Varshavsky 1984), both the IgH enhancer fragment and SPHI-oligonucleotide yielded identical tion of very low activity in LN-18 glioma cells (Figs. tissue-specific patterns with extracts from HeLa and 3-5, lane corePVUII; Table 1). This was unexpected, lymphoid (BJA-B)cell lines. Furthermore, the SPHI-oli- since mutagenesis of this protein-binding sequence re- gonucleotide competed with the IgH enhancer fragment duces activity of the complete SV40 enhancer when tested in HeLa cells (Zenke et al. 1986). for the same protein factor in BJA-B and HeLa cells (P. Matthias and W. Schaffner, unpubl.; see also Discus- In CV-1 cells the corePVUII-oligonucleotide stimu- sion). lates transcription to the same extent as the coreG-oli- In addition to the activity in lymphoid cells, a low gonucleotide (Fig. 3A, lanes coreC8x and corePVUII; stimulation of transcription by eight copies of the SPHI- Table 1). Thus, both oligonucleotides may contain se- segment was found in the nonlymphoid cell lines CV-1 quence motifs which are equally important for the ac- and LN-18 (Fig. 3A and D, lanes SPHI). It remains to be tivity of the complete SV40 enhancer in this cell line. determined whether the decanucleotide motif (which is Interestingly, the cell type specificities of the coreC- and probably responsible for activity of the SPHI-oligonu- the corePVUII-oligonucleotides are very different. While cleotide in lymphoid cells) or adjacent sequences, dis- coreC shows activity in a great variety of cells, the ac- tinct from those required in cells of lymphoid origin, are tivity of corePVUII is restricted to CV-1 cells. Since both responsible for this activity. contain the core consensus sequence GTGGA/TA/TA/ TG (though with one base difference in the A/T seg- ment; Fig. 1C, part b), it is likely that sequences next to Discussion the core consensus are responsible for the broad cell type specificity of the coreC element (see Discussion). Subsegments of the SV40 enhancer can act independently

SPHI-oligonucleotide shows lymphoid Our results show that multimers of short segments of cell-specific activity the SV40 enhancer contain all necessary sequence infor- mation for enhancer activity. Sequence motifs within The SPHI-oligonucleotide (25 bp} contains two direct re- these segments seem to be protein-binding sites (Da- peats of 9 bp of the sequence 5'AAGTATGCA/AAG- vidson et al. 1986; Wildeman et al. 1986)whose integrity CATGCA. These are essential for full activity of the is required for full activity of the SV40 enhancer (Herr complete SV40 enhancer in HeLa cells (Zenke et al. and Gluzman 1985; Herr and Clarke 1986; Zenke et al. 1986) and bind factors from HeLa cell extracts (Wil- 1986). We found that a high copy number of the indi- deman et al. 1986}. Interestingly, the junction of these vidual oligonucleotides is needed for significant activity direct repeats (ATGCA/AAGCA) is strongly homolo- (compare lanes coreC4x and coreG8x, Figs. 3 and 4). This gous (8/10 bp) to a motif that is conserved among immu- finding illustrates that a variety of different sequence noglobulin promoter and enhancer sequences (referred motifs, as they are present in natural enhancers, are not to as the decanucleotide; Falkner and Zachau 1984; required for the enhancer effect per se. There remains, Parslow et al. 1984). In another sequence context, this however, a strong requirement for multiple sequence

70 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Cell-type-specific SV40 enhancer segments motifs, even if they are of the same type, for optimal sequence corresponding to the decanucleotide is the enhancer activity. only homologous DNA stretch between the SPHI-oli- gonucleotide and the short IgH enhancer segment, these Broad cell type specificity of the coreC- but not the results suggest that the decanucleotide sequence is not core P V UII-oli g on u cl eo ti d e only responsible for the protein interaction seen in vitro but also for the in vivo activity of the SPHI-oligonucleo- The corePVUII-oligonucleotide corresponds to a subset tide in lymphoid cells. of nucleotides within the coreC-oligonucleotide with In agreement with this, the integrity of the decanu- only two mismatches. Both DNA segments contain a cleotide motif within the SV40 enhancer is of higher im- perfect match to the enhancer core consensus sequence portance in MPCll BU4 plasmacytoma cells than in GTGGA/TA/TA/TG (Fig. 1C, part b), which has been HeLa cells, as was shown by others (Davidson et al. shown to be important for SV40 enhancer activity 1986; Zenke et al. 1986): mutagenesis of this motif from (Weiher et al. 1983; Herr and Clarke 1986; Zenke et al. ATGCAAAGCA to ATTACAAGCA within the context 1986). Therefore, it is quite unexpected that these two of the complete enhancer reduced activity in MPC11 oligonucleotides display such radically different, though BU4 cells to 45% but only to 70% in HeLa cells. Con- overlapping, cell type specificities. versely, integrity of the 9-bp direct repeat seems to be Several hypotheses that are amenable to experimenta- more important for SV40 enhancer activity in HeLa tion could explain this observation. For example, the cells. In our construction, however, the 9-bp repeat ap- 20-bp-long coreG-oligonucleotide but not the shorter parently cannot contribute to enhancer activity in HeLa corePVUII-repeat might contain another protein-binding cells, which may be explained by a requirement for addi- site in addition to, or overlapping with, the core factor- tional flanking sequences, or by incorrect spacing of the binding site, a situation which could of course influence 9-bp repeat between tandem copies of the 25-bp-long cell type specificity. Alternatively, the core-binding pro- SPHI-oligonucleotide. tein could occur in multiple forms, either encoded by a family of related genes or as modified versions of one Interplay of sequence motifs in the context of the gene product. Different cell types would express a char- complete enhancer acteristic factor variant with a preferential affinity for a given core sequence. Furthermore, the affinity could A simple model accounting for the differential activity conceivably be modulated by the nature of the nucleo- of individual enhancer segments in various cell types as- tides adjacent to the consensus sequence motif and/or sumes that each segment binds a different factor de- the spacing between the motifs. Along this line of pending on the consensus motif contained within the thought, the factor variant of most cells would effi- segment and that the enhancer activity is then governed ciently bind to the coreC- but not the corePVUII-se- by the availability of the corresponding factor within a quence. The factor characteristic for CV-1 cells, how- given cell type. Consequently, an enhancer specific for a ever, could bind to both sequences. At the same time particular cell type would have to contain a combination this factor might have a relatively low affinity to the of sequence motifs for which binding factors are abun- coreC-sequence, since we note that, only in CV-1 cells, dant in that cell. A generally active enhancer could re- eight copies of the coreC-oligonucleotide are less active sult from a multitude of different sequence motifs, such than the SV40 enhancer (see Table 1). that inactivity of any given motif is balanced by the ac- tivity of others. We demonstrate that individual multimerized seg- Preferential activity of the SPHI-oligonucleotide in ments of the SV40 enhancer can act independently and lymphoid cells with a strict cell type specificity, divergent from each The SPHI-oligonucleotide contains a 9-bp direct repeat other and from the complete enhancer. Using synthetic whose junction forms the decanucleotide motif which is oligonucleotides provides the basis for gaining an under- also a typical component of immunoglobulin promoters standing of the function of individual enhancer motifs and enhancers (Falkner and Zachau 1984; Parslow et al. and their interaction in the context of the natural en- 1984). Interestingly, the cell type-specificity of the mul- hancer. However, it also appears from our investigations timerized SPHI-oligonucleotide correlates with that of a that, in its natural context, a given sequence is impor- 47-bp segment of the IgH enhancer which is also active tant for full enhancer activity in a greater variety of cell in lymphoid cells only. Both oligonuc|eotides contain types than after dissection. This seems to be the case for the decanucleotide motif, but the IgH enhancer segment the corePVUII- and SPHI-oligonucleotides (compare our lacks the 9-bp repeat present in the SV40 enhancer. data to those of Davidson et al. 1986; Herr and Clarke Within the IgH enhancer segment the decanucleotide 1986; Zenke et al. 1986). It is possible that these multi- motif plays a dominant role since its elimination results merized oligonucleotides are active by themselves only in both a severe downmutation and a loss of in vitro in certain cell lines that contain the cognate binding factor binding. In addition, the SPHI-oligonucleotide factors in high enough concentration or in a form of high competes in vitro with the IgH segment for the decanu- affinity. In the majority of cell lines, however, binding of cleotide-binding factor and both sequences show the the corePVUII- and SPHI-specific factors might require same cell type-specific behavior in bandshift assays (T. some cooperativity with neighboring factors, a coopera- Gerster, P. Matthias, and W. Schaffner, unpubl.). As the tivity that may be influenced by the spacing of sequence

GENES & DEVELOPMENT 71 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Schirm et al. motifs (Hochschild and Ptashne 1986; see also Taka- tandem. The EcoRI-XbaI fragment of this construct was then hashi et al. 1986; see below). Further investigations, blunt-ended and cloned into the blunt-ended XhoI-cleaved ex- such as mutagenesis and footprint analysis, will be re- pression vector JK- (Fig. 2A). The corresponding construct with quired to show the exact contribution of the sequence eight copies of the coreC-oligonucleotide was obtained by di- merization of the insert of the above clone. The tetramer-con- motifs to enhancer activity, and to exclude any influ- taining plasmid was cleaved on either side of the insert with ence of the junction sequence created by multimeriza- Asp-718 or BamHI, respectively, the protruding ends were re- tion of the oligonucleotides. Also, there may be differ- moved with S1 nuclease, and the resulting blunt ends were ently sized factor variants present in one or the other fused. A spacer of 4 bp was left between the two tetramer cell type. Thus, it will be interesting to elucidate copies. The corePVUII- and SPHI-oligonucleotides were multi- whether there is an effect on activity and cell type speci- merized and blunt-ended, and the gel-extracted material was ficity when varying the spacing between sequence cloned as octamers into the Sinai site of pUC18BA (Fig. 2B; motifs present in oligonucleotide repeats .... Yanisch-Perron et al. 1985); XhoI-SalI fragments from these Contrary to what might have been expected, our ex- constructs were then cloned into XhoI-cleaved JK- The orien- periments indicate that there is no obvious distinction tation of the enhancer segment within the expression vector is between classes of "constitutive" and "cell type-spe- the same for all constructs, as determined by Maxam-Gilbert sequencing (1980). cific" enhancers. Rather, a limited cell type specificity pUC18BA was constructed by replacing the BamHI-Asp 718 seems to be typical for individual enhancer segments. segment of the polylinker sequence in pUC18 (Yanisch-Perron Comparing the similar coreC- and corePVUII-oligonu- et al. 1985)by a double-stranded oligonucleotide, thus intro- cleotides, it appears that small variations in sequence ducing an additional restriction site for BglII and XhoI (Fig. 2B). and/or spacing of cis-acting regulatory elements can pro- As a negative-control plasmid for the transfection experi- foundly influence the cell type specificity. ments, polylinker sequences (XhoI-SalI segment of pUC18BA; Although we are only beginning to understand how Fig. 2B)were inserted into the XhoI site of JK-. enhancers of one or the other cell type specificity are For the construction of a reference gene with the human cy- assembled from combinations of different sequence tomegalovirus enhancer, a truncated rabbit ~-globin gene with 425 bp of 5'-flanking sequences (Picard and Schaffner 1985)was motifs, we should ultimately be able to construct en- introduced into pUC18BA. The enhancer-containing TaqI- hancers of desired cell type specificities by applying the HpaII segment of the SV40-HCMV recombinant C4 {Boshart et principle of combination and its underlying rules. Also, al. 1985) was filled in by Klenow polymerase and inserted into as already shown for the enhancer containing eight the SmaI site of pUC18BA upstream of the globin gene (Fig. copies of the coreC-oligonucleotide, such artificial en- 2C), resulting in a plasmid of about 7 kb. hancers can be considerably more active than their wild- type counterparts. Taken together, these findings should be of great value not only for basic research but also for DNA transfections therapeutic purposes involving cell type-specific effector Lymphoid cells were transfected by the DEAE-dextran protocol, delivery. as described by Luthman and Magnusson (1983), with modifica- tions according to de Villiers and Schaffner (1983) and Banerji et al. (1983). After removal of the DNA/DEAE-dextran mixture, the cells were treated with 25% DMSO (Picard and Schaffner Materials and methods 1985). Rat pituitary cells GH3 B6 were incubated for 45 min at Cell culture 37°C with 5 ml of medium without serum (100-ram plates), containing 20 ~g DNA of the test gene, 5 ~g of the reference Lymphoid cells Dll12, BJA-B, and Namalwa (gifts from U. gene, and 200 ~g/ml DEAE-dextran (F. Pasleau, pers. comm.). Weidle, Tutzing; H. zur Hausen, Heidelberg; C. Weissmann, Transfections of the other adherent cell lines by the calcium Ziirich, respectively)were grown in RPMI 1640 medium sup- phosphate coprecipitation followed the protocol by Graham plemented with 10% fetal calf serum. For Dl112 cells, the me- and van der Eb (1973) and Wigler et al. (1978), with modifica- dium contained, in addition, 1 rnM sodium pyruvate, 2 m~ L- tions described by Weber et al. (1984). For each transfection the glutamine and 1% nonessential amino acids. The other cell test gene (8.5 kb) and the reference gene (8.2 kb)were used in lines were propagated in Dulbecco's modified Eagles minimal the molar ratio 4:1 (except for the transfection of GH3 B6 medium (Gibco). For HeLa and CV-1 cells, the medium con- where test and reference gene were used in the molar ratio tained 2.5% calf serum and 2.5% fetal calf serum; for 3T6, 3:1). MCF7, LN-18, Molt-4, GH3 B6, and Bowes (gifts from W. Topp; G. Schiitz, Heidelberg; N. de Tribolet, Lausanne; B. Flecken- stein, Erlangen; A. Belayew, Liege; J. Briiggen, Basel, respec- RNA analysis tively), the medium contained 10% fetal calf serum. Media were supplemented with 100 U/ml penicillin and 100 ~g/ml Total cytoplasmic RNA was extracted 45 hr after transfection streptomycin. The cells were seeded 1 day before transfection. {de Villiers and Schaffner 1983) and digested with proteinase K, At the time of transfection, 3T6 cells were 20-30% confluent, and residual input plasmid DNA was removed by RNase-free the other adherent cells 50-70%. Lymphoid cells (suspension DNase (Picard and Schaffner 1983). $1 nuclease mapping was cultures) had a density of 2 x 106 to 5 x 106 cells/ml (in 100- performed with a single-stranded DNA probe labeled at the mm plates containing 10 ml of medium). BamHI site [position 480) of a rabbit B-globin gene (Rusconi and Schaffner 1981) as described in Weaver and Weissmann {1979) and de Villiers and Schaffner (1983). Depending on the cell line, Construction of plasmids between 10 and 100 ~g of RNA were used for each analysis. A tetramer of the coreC-oligonucleotide was cloned into the (The level of transiently expressed f~-globin-specific RNA was SmaI site of pSVXB (Tognoni et al. 1985) as a head-to-tail generally low for lymphoid cells and GH3 B6 cells.}

72 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Cell-type-specific SV40 enhancer segments

Acknowledgments Kamen. 1984. Polyoma virus DNA replication requires an enhancer. Nature 312: 242-246. We are especially grateful to Hans Rink (Ciba Geigy, Basel, Di- Dorsch-H~isler, K., G.M. Keil, F. Weber, M. Jasin, W. Schaffner, vision of Biotechnology) for synthesizing the coreC-oligonu- and U.H. Koszinowski. 1985. A long and complex enhancer cleotide and further oligonucleotides that were investigated in activates transcription of the gene coding for the highly some initial experiments. We also thank Alexandra Belayew abundant immediate early mRNA in murine cytomegalo- (Universite de Liege), Sart Tilman and Winship Herr (Cold virus. Proc. Natl. Acad. Sci. 82: 8325-8329. Spring Harbor Laboratory, Cold Spring Harbor) for communica- Falkner, F.G. and H.G. Zachau. 1984. Correct transcription of tion of results prior to publication, the colleagues mentioned in an immunoglobulin kappa gene requires an upstream frag- the text for gifts of cell lines, Wemer Ziircher (Friedrich ment containing conserved sequence elements. Nature Miescher Institute, Basel) for help with oligonucleotide syn- 310: 71-74. thesis, Fritz Ochsenbein for the preparation of the figures, and Gillies, S.D., S.L. Morrison, V.T. Oi, and S. Tonegawa. 1983. A Jutta Buxtorf for help in typing the manuscript. Finally, we ac- tissue specific transcription enhancer element is located in knowledge Dani Schiimperli, Fred Schaufele, and Charles E. the major intron of a rearranged immunoglobulin heavy Samuel for critical reading of the manuscript. This work was chain gene. Cell 33:717- 728. supported by Schweizerischer Nationalfonds and the Kanton Gluzman, Y. 1985. Eukaryotic transcription. The role of cis- Ztirich. S.S. was the recipient of an EMBO fellowship. and trans-acting elements in initiation. In Current Commu- nications in Molecular Biology (ed. Y. Gluzman). Cold Note added in proof Spring Harbor Laboratory, Cold Spring Harbor, New York. Gorrnan, C., L. Moffat, and B. Howard. 1982. Recombinant After submission of our manuscript, we have learned that W. genomes which express chloramphenicol acetyltransferase Herr and his colleagues, in independent experiments, have ob- in mammalian cells. Mol. Cell. Biol. 2: 1044-1051. served different cell type specificities of individually polymer- Graham, F.L. and A.I. van der Eb. 1973. A new technique for the ized segments of the SV40 enhancer (B. Ondek, A. Shepard, and assay of infectivity of human adenovirus 5 DNA. Virology W. Herr, EMBO J., submitted). 52: 456-467. Harvey, R.P., A.I. Robins, and J.R.E. Wells. 1982. Independently References evolving chicken histone H2B genes: Identification of a ubiquitous H2B-specific 5' element. Nucleic Acids Res. Amati, P. 1985. Polyoma regulatory region: A potential probe 10: 7851-7863. for mouse cell differentiation. Cell 43:561-562. Herbomel, P., B. Bourachot, and M. Yaniv. 1984. Two distinct Banerji, J., S. Rusconi, and W. Schaffner. 1981. Expression of a enhancers with different cell specificities coexist in the reg- beta-globin gene is enhanced by remote SV40 DNA se- ulatory region of polyoma. Cell 39: 653-662. quences. Cell 27: 299-308. Herr, W. and I. Clarke. 1986. The SV40 enhancer is composed of Banerji, J., L. Olson, and W. Schaffner. 1983. A lymphocyte-spe- multiple functional elements that can compensate for one cific cellular enhancer is located downstream of the joining another. Cell 45: 461-470. region in immunoglobulin heavy chain genes. Cell 33: 729- Herr, W. and Y. Gluzman. 1985. Duplications of a mutated 740. simian virus 40 enhancer restore its activity. Nature Boshart, M., F. Weber, G. Jahn, K. Dorsch-H~isler, B. Flecken- 313: 711-714. stein, and W. Schaffner. 1985. A very strong enhancer is lo- Hochschild, A. and M. Ptashne. 1986. Cooperative binding of cated upstream of an immediate early gene of human cyto- lambda repressors to sites separated by integral turns of the megalovirus. Cell 41: 521-530. DNA helix. Cell 44: 681-687. Buchman, A.R., L. Bumett, and P. Berg. 1981. The SV40 nu- Laimins, L.A., G. Khoury, C. Gorman, B. Howard, and P. Gruss. cleotide sequence. In Molecular biology of tumor , 1982. Host-specific activation of transcription by tandem 2nd edition, revised: DNA tumor viruses. (ed. J. Tooze), pp. repeats from simian virus 40 and Moloney murine sarcoma 799-841. Cold Spring Harbor Laboratory, Cold Spring virus. Proc. Natl. Acad. Sci. 79: 6453--6457. Harbor, New York. Laimins, L.A., P. Tsichlis, and G. Khoury. 1984. Multiple en- Camper, S.A., Y.A.S. Yao, and F.M. Rottman. 1985. Hormonal hancer domains in the 3' terminus of the Prague strain of regulation of the bovine prolactin promoter in rat pituitary Rous sarcoma virus. Nucleic Acids Res. 12: 6427-6442. tumor cells. J. Biol. Chem. 260: 12246-12251. Luthman, H. and G. Magnusson. 1983. High efficiency polyoma Davidson, I., C. Fromental, P. Augereau, A. Wildeman, M. DNA tranfection of chloroquine treated cells. Nucleic Acids Zenke, and P. Chambon. 1986. Cell-type specific protein Res. 11: 1295-1308. binding to the enhancer of simian virus 40 in nuclear ex- Mattaj, I.W., S. Lienhard, J. liricny, and E.M. De Robertis. 1985. tracts. Nature 323: 544-548. An enhancer-like sequence within the Xenopus U2 gene de Villiers, I. and W. Schaffner. 1981. A small segment of promoter facilitates the formation of stable transcription polyoma virus DNA enhances the expression of a cloned complexes. Nature 316: 163-167. beta-globin gene over a distance of 1400 base pairs. Nucleic Maxam, A.M. and W. Gilbert. 1980. Sequencing end-labelled Acids Res. 9: 6251-6264. DNA with base-specific chemical cleavages. Methods En- ~.1983. Transcriptional "enhancers" from papovaviruses zymol. 65: 499-560. as components of eukaryotic expression vectors. In Tech- Moreau, P., R. Hen, B. Wasylyk, R. Everett, M.P. Gaub, and P. niques in nucleic acid biochemistry (B5)(ed. R.A. Flavell), Chambon. 1981. The SV40 72 base repair repeat has a pp. B507/1-B507/20. Elsevier, Amsterdam. striking effect on both in SV40 and other de Villiers, J., L. Olson, C. Tyndall, and W. Schaffner. 1982. chimeric recombinants. Nucleic Acids Res. 9: 6047-6068. Transcriptional "enhancers" from SV40 and polyoma virus Neuberger, M.S. 1983. Expression and regulation of immuno- show a cell type preference. Nucleic Acids Res. 10: 7965- globulin heavy chain gene transfected into lymphoid cells. 7976. EMBO ]. 2: 1373-1378. de Villiers, J., W. Schaffner, C. Tyndall, S. Lupton, and R. Neuhaus, G., G. Neuhaus-Url, P. Gruss, and H.G. Schweiger.

GENES & DEVELOPMENT 73 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

Schinn et al.

1984. Enhancer-controlled expression of the simian virus 40 Wildeman, A.G., M. Zenke, C. Schatz, M. Wintzerith, T. T-antigen in the green alga Acetabularia. EMBO J. 3: 2169- Grundstr6m, H. Matthes, K. Takahashi, and P. Chambon. 2172. 1986. Specific protein binding to the simian virus 40 en- Parslow, T.G., D.L. Blair, W.J. Murphy, and D.K. Granner. 1984. hancer in vitro. Mol. Cell Biol. 6: 2098-2105. Structure of the 5' ends of immunoglobulin genes: A novel Yanisch-Perron, C., ]. Vieira, and ]. Messing. 1985. Improved conserved sequence. Proc. Natl. Acad. Sci. 81: 2650-2654. M13 phage cloning vectors and host strains: Nucleotide se- Picard, D. and W. Schaffner. 1983. Correct transcription of a quences of the M13mpl8 and pUC19 vectors. Gene cloned mouse immunoglobulin gene in vivo. Proc. Natl. 33: 103-119. Acad. Sci. 80: 417-421. Zenke, M., T. Grundstr6m, H. Matthes, M. Wintzerith, C. ~.1985. Cell-type preference of immunoglobulin kappa Schatz, A. Wildeman, and P. Chambon. 1986. Multiple se- and lambda gene promoters. EMBO J. 4:2831-2938. quence motifs are involved in SV40 enhancer function. Rusconi, S. and W. Schaffner. 1981. Transformation of frog em- EMBO J. 5: 387-397. bryos with a rabbit beta-globin gene. Proc. Natl. Acad. Sci. 78:5051-5055. Schirm, S., F. Weber, W. Schaffner, and B. Fleckenstein. 1985. A transcription enhancer in the Herpesvirus saimiri genome. EMBO J. 4: 2669-2674. Sch61er H.R. and P. Gruss, 1984. Specific interaction between enhancer-containing molecules and cellular components. Cell 36:403-411. Sen, R. and D. Baltimore. 1986. Multiple nuclear factors in- teract with the immunoglobulin enhancer sequences. Cell 46: 705- 716. Serfling, E., M. Jasin, and W. Schaffner. 1985. Enhancers and eukaryotic gene transcription. Trends Genet. l: 224-230. Sive, H.L., N. Heintz, and R.G. Roeder. 1986. Multiple se- quence elements are required for maximal in vitro tran- scription of a human histone H2B gene. Mol. Cell. Biol. 6: 3329-3340. Strauss, F. and A. Varshavsky. 1984. A protein binds to a satel- lite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome. Cell 37: 889-901. Swimmer, C. and T. Shenk. 1984. A viable simian virus 40 variant that carries a newly generated sequence reiteration in place of the normal duplicated enhancer element. Proc. Natl. Acad. Sci. 81: 6652-6656. Takahashi, K., M. Vigneron, H. Matthes, A. Wildeman, M. Zenke, and P. Chambon. 1986. Requirement of stereospe- cific alignments for initiation from the simian virus 40 early promoter. Nature 319: 121-126. Tognoni, A., R. Cattaneo, E. Serfling, and W. Schaffner. 1985. A novel expression selection approach allows precise mapping of the virus enhancer. Nucleic Acids Res. 13: 7457-7472. Veldman, G.M., S. Lupton, and R. Kamen. 1985. Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression. Mol. Cell. Biol. 5: 649-658. Weaver, R.F. and C. Weissmann. 1979. Mapping of a RNA by a modification of the Berk-Sharp procedure: The 5' termini of 155 beta globin mRNA precursor and mature 10S beta globin mRNA have identical map coordinates. Nucleic Acids Res. 7:1175-1193. Weber, F. and W. Schaffner. 1985. Enhancer activity correlates with the oncogenic potential of avian retroviruses. EMBO ]. 4: 949-956. Weber, F., J. de Vflliers, and W. Schaffner. 1984. An SV40 "en- hancer trap" incorporates exogenous enhancers or generates enhancers from its own sequences. Cell 36: 983-992. Weiher, H., M. K6nig, and P. Gruss. 1983. Multiple point muta- tions affecting the simian virus 40 enhancer. Science 219:626-631. Wiglet, M., A. Pellicer, S. Silverstein, and R. Axel. 1978. Bio- chemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell 14: 725-731.

74 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press

The SV40 enhancer can be dissected into multiple segments, each with a different cell type specificity.

S Schirm, J Jiricny and W Schaffner

Genes Dev. 1987, 1: Access the most recent version at doi:10.1101/gad.1.1.65

References This article cites 49 articles, 12 of which can be accessed free at: http://genesdev.cshlp.org/content/1/1/65.full.html#ref-list-1

License

Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the top Service right corner of the article or click here.

Copyright © Cold Spring Harbor Laboratory Press